Mouse igg1 pe

IL-2R consists of three receptors: CD25, CD122, and CD132. To measure the expression of these three receptors in C8166 cells, C8166 cells (1 × 10⁶ cells/mL) in 24-well plates were first harvested and washed twice with 1% BSA/PBS, resuspended with 1 mL of 1% BSA/PBS, and co-incubated for 60 min at 4 °C with 20 μg of FITC anti-CD25 antibody (Cat No.: 11025742, Thermo Fisher, USA), PE anti-CD122 antibody (Cat No.: 46122842, Thermo Fisher, USA), PE anti-CD132 antibody (Cat No.: 12132942, Thermo Fisher, USA) or the corresponding isotype control (Mouse IgG2b-FITC, CatNo.: 11473281; Mouse IgG1-PE, Cat No.: 46471482; Rat IgG2b-PE, Cat No.: 12403182, Thermo Fisher, USA), respectively.
To measure STAT5 phosphorylation, 1 × 10⁶ C8166 cells for each test in 24-well plates were harvested and stimulated for 60 min at 37 °C with 10 ng doses of IL-2. The samples were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer Set (Cat No.: 88882400, Thermo Fisher, USA) following the manufacturer's instructions and incubated for 60 min in 4 °C in different doses of PE anti-phospho-STAT5 (Tyr694) antibody (Cat No.: 12901042, Thermo Fisher, USA) and isotype control (Cat No.: 12471482, Thermo Fisher, USA), respectively. Finally, the cells were measured using a flow cytometer (BD FACSCalibur) and analyzed with FlowJo software (Flow Jo LLC, Ashland, OR, USA).